JP2002539223A - Human sperm surface antigen - Google Patents

Abstract

  (57) [Summary] The present invention relates to sperm surface proteins and the use of such proteins to regulate fertility in mammalian species. More particularly, the present invention relates to purified sperm-specific surface peptide epitopes and derivatives of those protein epitopes and antibodies to those protein epitopes. The present invention relates to therapeutic and diagnostic compositions in procedures related to infertility and methods of using those compositions. The compositions of the present invention comprise a sperm cell surface antigen recognized by anti-sperm antibodies present in infertile male and female sera. The invention further relates to therapeutic compositions and methods of diagnosis and treatment. These include compositions and methods that modulate (ie, inhibit or promote) infertility.

Description

DETAILED DESCRIPTION OF THE INVENTION

This invention was made with United States Government support under grant numbers HD-29099, P30-28934, and D543-TW00655, awarded by the National Institutes of Health. . The United States Government has certain rights in the invention.

FIELD OF THE INVENTION [0002] The present invention relates to sperm cell surface antigens. This antigen is recognized by anti-sperm antibodies present in infertile male and female sera. More particularly, the present invention relates to epitopes on sperm-specific surface proteins / peptides and derivatives and antibodies directed against those protein epitopes.

BACKGROUND OF THE INVENTION [0003] The correlation of anti-sperm antibodies (ASA) with some cases of unexplained infertility suggests a role for these antibodies in blocking fertilization. The incidence of immunity to sperm in infertile couples is estimated to be 9-36%.
In contrast, the transmission of ASA in the general population is approximately 0-2%. Anti-sperm antibodies are thought to impair fertility by inhibiting sperm motility, sperm penetration of cervical mucus, capacitation or acrosome, or anti-sperm antibodies trigger the complement cascade, It can result in sperm lysis.

[0004] A thorough understanding of the mechanisms behind immunological infertility and improved diagnosis and treatment rely on the knowledge of the identification of specific sperm antigens that can trigger the production of functionally related sperm antibodies. For example, if a recombinant sperm surface antigen is available, that antigen can serve as a target for an immunoassay in patient sera that detects the presence of certain antibodies that mediate infertility. In addition, anti-sperm antibodies and their cognate antibodies, in the form of a birth control vaccine, may provide the basis for immunological control of infertility.

[0005] Several studies have reported the identification of sperm antigens recognized by systemic and / or local autoantibodies and isoantibodies from infertile individuals using immunoblot techniques. However, in most cases, the reaction between sperm antigens and control sera from fertile individuals has not been described, and the functional relevance of these antibodies in fertility remains unclear. In addition, most of the earlier reports used one-dimensional gel electrophoresis for the separation of sperm proteins.

[0006] The present invention relates to proteins identified by high resolution 2D electrophoresis.
The sperm antigen is separated in the first dimension (screening for acidic and basic proteins) by either isoelectric focusing or non-equilibrium pH gradient electrophoresis, followed by PAGE and sensitive western blot. Further, the sample serum was subjected to an immunobead test (IBT, Bronson R, Cooper G, R
osenfeld D. , Jagiello G and VogueI H (ed.), Bioregulators of reproduction, acad
e.m., press, New York 1981: 521-527), and utilizing only sera that show significant reactivity for the presence of anti-sperm antibodies, utilizing immunodominance. The antigen was identified. In addition, sperm antigens unique to infertile patients were identified by excluding antigens recognized by serum from clinically infertile subjects using computer comparison of 2D immunoblots. Database of 2D gel images of silver-stained proteins (sperm proteome) and databases of vectorically labeled sperm surface proteins (sperm surface index, Biol Reprod 199)
7; 56: 771-787) allowed the definition of a subset of sperm surface antigens associated with antibody-mediated infertility.

SUMMARY OF THE INVENTION The present invention relates to therapeutic and diagnostic compositions in procedures related to infertility and methods of using those compositions. The compositions of the present invention comprise a sperm cell surface antigen recognized by anti-sperm antibodies present in infertile male and female sera. The invention further relates to therapeutic compositions and methods of diagnosis and treatment. These include compositions and methods that modulate (ie, inhibit or promote) infertility.

DETAILED DESCRIPTION OF THE INVENTION In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, “nucleic acid”, “DNA” and similar terms also include
Nucleic acid analogs (ie, analogs having other than phosphodiester backbones)
Is included. For example, so-called "peptide nucleic acids", which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, are considered within the scope of the invention.

[0010] As used herein, "effective amount" means an amount sufficient to produce a selected effect. For example, an effective immunizing amount of a sperm surface protein is an amount sufficient to elicit an immune response in a host to which the vaccine preparation is administered.

As used herein, the term “pharmaceutically acceptable” is approved by a federal or national governmental regulatory authority, or in the United States Pharmacopeia or animals (more specifically, humans). ) Means listed in other generally recognized pharmacopeias.

As used herein, the term “carrier” refers to a diluent, adjuvant,
Excipient or vehicle with which the therapeutic is administered.

[0013] The present invention relates to sperm surface antigens that are recognized by serum isolated from infertile patients, but are not recognized by serum isolated from clinically fertile subjects. The sperm antigens of the present invention are unique to infertile patients and are
Immunoblot comparisons were used to identify antigens by clinically excluding antigens recognized by serum from infertile subjects. Database of 2D gel images of silver stained proteins (sperm proteome) and databases of vectorically labeled sperm surface proteins (sperm surface index, Biol Reprod)
1997; 56: 771-787) allowed the definition of a subset of sperm surface antigens associated with antibody-mediated infertility. These identified sperm surface proteins are referred to herein genetically as SSPs and include isolated native proteins and fragments thereof as well as recombinant derivatives and synthetic analogs of the native protein.

[0014] SSP proteins, polypeptides and peptide fragments thereof can be prepared for various uses. For example, such molecules may be used for the production of antibodies, for use in diagnostic and therapeutic assays, for the identification of other sperm gene products involved in sperm motility, or for the identification of compounds that modulate sperm motility. Can be used for

[0015] One embodiment of the invention is directed to a pharmaceutical composition comprising a sperm surface peptide epitope and a pharmaceutically acceptable carrier. The pharmaceutical carrier may contain sterile liquids, such as water and oils (oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.) and buffered saline, dextrose, water, Glycerol, including sterile isotonic aqueous buffer) and combinations thereof)
Can be Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Suitable pharmaceutical excipients for use in the compositions of the present invention include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, Glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like can be mentioned. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions include solutions, suspensions, emulsions, tablets, pills, capsules, powders,
It may take the form of a sustained release formulation or the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as the following: pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are
Remington's Pharmaceutical Sciences "
E. FIG. W. It is described in Martin. Such compositions comprise a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier, thereby providing the patient with a form for proper administration. The formulation should suit the mode of administration.

According to one embodiment, an SSP comprising a composition of the invention is used to modulate fertility in a mammalian species. The method comprises administering a composition comprising a therapeutically effective amount of an SSP antigen. In one preferred embodiment, the composition is formulated as a contraceptive vaccine comprising one or more SSPs (or SSP protein fragments or analogs thereof) and is administered to a mammalian species to elicit an immune response.

In one embodiment, the SSP-containing composition is used to treat a subject for a fertility-related disorder by administering an effective amount of a sperm surface protein or fragment thereof. In particular, the SSP compositions of the present invention can be used in a method for treating immunological infertility. The method comprises obtaining a serum sample from an individual exhibiting immunological infertility, determining a sperm surface protein targeted by antibodies present in the individual's serum, and targeted by the antibody of the individual. One or more peptides (or peptide derivatives or peptidomimetics)
Administering a composition comprising: In one embodiment, the composition is administered locally to the female to bind to the individual's anti-sperm antibody and, thus, enhance the female's fertility.

According to another embodiment, the SSPs of the invention can be used to screen for molecules that interact with sperm surface proteins, including those that bind surface proteins. Contacting one or more molecules with the sperm-specific protein / peptide under conditions that permit complex formation between the sperm-specific protein / peptide and the molecule; Recovering a molecule that specifically binds to the sperm surface protein / peptide.

According to one embodiment, the present invention provides polypeptides and their respective fragments and analogs, having at least one of the following activities: may modulate the ability of sperm to fertilize eggs. That it functions as a sperm marker, has the ability to bind to serum isolated from an infertile individual, and is immunogenic. In one preferred embodiment, the sperm surface protein is a human protein comprising an amino acid sequence selected from the group consisting of: Ile Asn Ser Gln Trp Val Val Pro Leu
Arg (SEQ ID NO: 1); Leu Asn Ser Gln Trp Val Val Pro Leu
Arg (SEQ ID NO: 2); Met Val Asn Ile Met Ile Ile Ile Arg (
SEQ ID NO: 3); Met Val Asn Leu Met Ile Ile Ile Arg (
SEQ ID NO: 4); Met Val Asn Ile Met Leu Ile Ile Arg (
SEQ ID NO: 5); Met Val Asn Ile Met Ile Leu Ile Arg (
SEQ ID NO: 6); Met Val Asn Ile Met Ile Ile Leu Arg (
SEQ ID NO: 7); Met Val Asn Ile Met Ile Leu Leu Arg (
SEQ ID NO: 8); Met Val Asn Ile Met Leu Leu Ile Arg (
SEQ ID NO: 9); Met Val Asn Ile Met Leu Ile Leu Arg (
SEQ ID NO: 10); Met Val Asn Leu Met Leu Ile Ile Arg (
SEQ ID NO: 11); Met Val Asn Leu Met Leu Ile Ile Arg (
SEQ ID NO: 12); Met Val Asn Leu Met Ile Leu Ile Arg (
SEQ ID NO: 13); Met Val Asn Leu Met Ile Ile Leu Arg (
SEQ ID NO: 14); Met Val Asn Leu Met Ile Leu Leu Arg (
SEQ ID NO: 15); Met Val Asn Leu Met Leu Leu Ile Arg (
SEQ ID NO: 16); Met Val Asn Leu Met Leu Ile Leu Arg (
Met Ile Pro Val Ile Glu (SEQ ID NO: 18); Met Leu Pro Val Ile Glu (SEQ ID NO: 19); Met Ile Pro Val Leu Glu (SEQ ID NO: 20); Met Leu Pro Val Val (SEQ ID NO: 18) 21); His Gly Gln Ser Ala Glu Tyr Glu Phe (
SEQ ID NO: 22); and Phe Gln Gln Asp Gly Gly Ala Ser (SEQ ID NO: 23).

In one aspect of the invention, a composition comprising a sperm surface peptide epitope of the invention is administered as a contraceptive vaccine formulation. More specifically, the sperm surface peptide epitope is an isolated or recombinant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23. In one embodiment, the epitope is a peptide consisting of a sequence selected from SEQ ID NOs: 1-23.

According to one embodiment, the invention provides a method for modulating fertility in a mammalian species. The method comprises administering a composition comprising a therapeutically effective amount of an isolated or recombinant polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23. And a step.

Alternatively, the composition may include a fragment of the polypeptide or the polypeptide. This polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23. In one embodiment, the composition comprises a peptide having a sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23.

[0024] In one embodiment, a composition for modulating fertility in a mammalian species comprises a polypeptide antigen. The polypeptide antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. In another embodiment, the composition comprises a polypeptide antigen. This polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-17. In another embodiment, the composition comprises a polypeptide antigen. This polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-21. In another embodiment, the composition comprises
Includes polypeptide antigens. This polypeptide comprises the amino acid sequence of SEQ ID NO: 22. In another embodiment, the composition comprises a polypeptide antigen. This polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

[0025] The present invention also encompasses nucleic acid sequences encoding the peptide sequences represented by SEQ ID NOs: 1-23. Nucleic acid sequences encoding these peptides can be synthesized using standard techniques known to those skilled in the art. In addition, including these amino acid sequences,
Gene sequences encoding full-length polypeptides can be isolated from eukaryotic genomes, including mammalian genomes, using standard techniques known to those of skill in the art. In a preferred embodiment, the nucleic acid sequence encoding the SSP is from the human genome.

The SSPs of the present invention are proteins that represent derivatives of the native protein (ie,
Deletions, including internal deletions, additions resulting in fusion proteins, of the amino acid sequences within and / or adjacent to the above amino acid sequences (including SEQ ID NOs: 1-23);
Or proteins that contain substitutions (which result in “silent” changes, which produce functionally equivalent peptides). Amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the relevant residues. For example, non-polar (hydrophobic) amino acids include
These include: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids include: glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; positively charged (Basic) amino acids include: arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Alternatively, if a change in function is desired, a deletion or non-conservative change may
It can be engineered into a nucleic acid sequence encoding P to produce an altered SSP gene product. In addition, such alterations can be selected to produce a product that is better suited for expression, scale-up, etc., in the selected host cell. For example,
In one embodiment, a cysteine residue may be deleted or replaced with another amino acid residue to remove a disulfide bridge. In another embodiment, a tyrosine residue may be deleted or replaced with another amino acid residue to remove tyrosine phosphorylation.

The SSP protein or a part of the SSP protein (eg, SEQ ID NOS: 1 to 23)
Are also within the scope of the present invention. Such proteins and peptides are shown in SEQ ID NOs: 1-2.
3 can be designed based on a synthetic nucleotide sequence encoding the amino acid sequence.
Fusion proteins include, but are not limited to: an IgFc fusion that stabilizes the SSP protein or peptide and enhances its half-life in vivo; or allows the fusion protein to be anchored to the cell membrane A fusion to any amino acid sequence; or a fusion of an SSP protein or peptide to an enzyme, fluorescent protein, luminescent protein or flag epitope protein or peptide that provides a marker function.

[0029] The SSP polypeptides of the invention may further include post-translational modifications, including, but not limited to: stearylation, myristylation, palmitation, glycosylation, acetylation, and phosphorylation. If the native SSP does not have a recognition motif that allows for such modification, one of skill in the art can introduce an SSP gene nucleotide sequence that encodes a motif such as an enzyme recognition signal to generate an SSP gene product. Is routine for those skilled in the art.

The present invention also encompasses the synthesis of peptides and peptide analogs of the native protein and peptide sequences disclosed herein. Such peptides or analogs thereof can be prepared using virtually any technique known in the art for the preparation of peptides and peptide analogs. For example, the peptide can be prepared in linear form using conventional solution or solid phase peptide synthesis, and cleaved from the resin, followed by a purification procedure (Creight
on, 1983, Protein Structures And Molec
ullar Principles, W.C. H. Freeman and Co. ,
N. Y. ). Suitable procedures for synthesizing the peptides described herein are well known in the art. The composition of the synthetic peptide can be confirmed by sequencing amino acid analysis (eg, Edman degradation procedure and mass spectrometry).

In addition, analogs and derivatives of the peptides can be chemically synthesized. The linkage between each amino acid of the peptides of the invention can be an amide, a substituted amide or an isoelectronic configuration of an amide. Non-classical amino acids or chemical analogs of amino acids can be introduced as a substitution or addition to the sequence. Non-classical amino acids include, but are not limited to: D isomers of common amino acids, α-
Aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-
Abu, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, Cyclohexylalanine, (3-alanine, fluoro-amino acids, designer amino acids (eg, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general) The amino acid is D (dextrorotatory) ) Or L (Levorotary).

[0032] Cyclized peptides can be formed by the addition of Cys residues to the termini of linear peptides. Disulfide bond formation, if desired, is generally performed in the presence of a mild oxidizing agent. A chemical oxidant may be used, or the compound may simply be exposed to atmospheric oxygen to effect these bonds. Various methods are known, including, for example, those described by: Tam
, J. et al. P. Et al., 1979, Synthesis 955-957; Stewar.
t et al., 1984, Solid Phase Peptide Synthesis.
, Volume 2, Pierce Chemical Company Rockfor
d, IL; Ahmed et al., 1975, J. Am. Bioi. Chem. 250: 84
77-8482; and Pennington et al., 1991 Peptide.
s 1990 164-166, Giralt and Andreu, Eds.
, ESCOM Leiden, The Netherlands. Further alternatives are described by: Kamber et al., 1980, Helv Chim-.
Acta 63: 899-915. The method performed on a solid support is described by: Albericio, 1985, Int. -J. Peptid
e Protein Res. 26: 92-97. Any of these methods can be used to form disulfide bonds in the peptides of the invention.

If the amino acid (eg, SEQ ID NOs: 1-23) that the peptide is genetically encoded is made up of entirely, or if that portion is so made up, then the peptide or related portion thereof will be It can be synthesized using recombinant genetic engineering techniques. For recombinant production, a polynucleotide sequence encoding the linear form of the peptide can be synthesized and a suitable expression vehicle (ie, a vector containing the necessary elements for transcription and translation of the inserted coding sequence, or R
In the case of a NA virus vector, it can be inserted into a vector containing elements necessary for replication and translation). The expression vehicle is then transfected into a suitable target cell. The cell expresses the peptide. The expressed peptide is then isolated by procedures well established in the art, depending on the expression system used. Methods for the production of recombinant proteins and peptides are well known in the art (see, eg, Mania
tis et al., 1989, Molecular Cloning A Labora.
tory Manual, Cold Spring Harbor Labor
atory, N .; Y. And Ausubel et al., 1989, Current.
Protocols in Molecular Biology, Green
e Publishing Associates and Wiley In
terscience, NY). Methods for introducing codon substitutions into a native sequence to encode an antagonistic peptide based on the disclosure herein are well known to those skilled in the art. The SSP gene product, its peptide fragments and its fusion proteins can be produced by recombinant DNA technology using techniques well known in the art.

[0034] According to one embodiment, the SSP of the present invention may comprise a fertility-related disorder associated with the presence of an antibody directed against a sperm surface protein, or a predisposition for developing the disorder. , Diagnostic or screening methods. More specifically, in one embodiment, the method is used to diagnose immunological infertility by detecting the presence of an antibody against a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-23. Or screen. The method comprises obtaining a blood sample from an individual, isolating the serum, contacting the serum sample with a composition comprising a sperm surface antigen, and specifically binding to the sperm surface antigen. Determining the presence of the antibody in the serum sample. The presence of such antibodies indicates the presence of a predisposition to a disorder associated with fertility.

Detection of antibodies in serum samples that specifically bind to sperm surface antigen
It can be performed using standard techniques known to those skilled in the art, such as mobility shift assays and the use of labeled components. For example, the SSP can be covalently attached to an inert solid support and contacted with the sample serum. Then, the S
The SP is washed to remove any unbound and non-specifically bound serum components. The SSP is then labeled with a labeled secondary antibody specific for the antibody of the organism that provided the serum sample (ie, if the serum sample is human, the front-ended secondary antibody is (Which is a human antibody).

[0036] The SSP can be bound to a variety of solid supports, such as agarose, Sepharose, polystyrene or other chromatographic beads, using protocols available to those skilled in the art. For example, a primary amine can be conjugated to NHS, cyanogen bromide activated resin or maleimide activated resin. Many of these resins are commercially available (eg, Pharmacia Corporation).
'Activated Sepharose). Alternatively, SSP can be reacted through its carboxylic acid residue, for example, with the commercially available resin Pharmacia EAH-activated Sepharo.
Connected by se.

According to one embodiment of the present invention, the sperm surface proteins of the present invention are used as vaccines in human and non-human animals. More specifically, the present invention involves the use of SSP-based vaccines for contraception. In one aspect of the invention, SSP may be delivered to a subject to elicit an active immune response and act as an antagonist of temporary or reversal of conception. For example, such vaccines can be used for active immunization of a subject to elicit an antibody response that temporarily blocks sperm motility or access to eggs. In one aspect of the invention, the antigen may be administered for a specific period of the month, for example, during the ovulation phase, to elicit anti-sperm antibody titers in a female subject to block fertility.

In another embodiment of the present invention, the SSP is useful as a vaccine for permanent sterilization of a subject. Such a vaccine can be used to elicit a T cell-mediated attack on sperm cells, which is useful as a method for irreversible sterilization.
Methods for generating T cell-specific responses (eg, adoptive immunotherapy) are well known in the art (see, eg, Vaccine Design).
n, Michael F. Powell and Mark J. Newman
Eds. , Plenum Press, New York, 1995, pp.
847-867). Such techniques may be particularly useful for veterinary contraception or sterilization purposes. In this case, a single dose vaccination may be desired.

[0039] Sperm surface protein antigens can be produced in large quantities and purified for use in vaccine preparations. The sperm surface proteins of the invention also have the following utility in immunoassays: for example, detecting or measuring the presence of antibodies for the antigen in a sample of body fluid from a vaccinated subject; And thus diagnosing and / or monitoring the immune response of the subject, followed by vaccination. Vaccine preparations containing an immunogenic polypeptide as an active ingredient are known to those of skill in the art (see, for example, Vaccine Design, Michael F. Powell an).
d Mark J. et al. Newman Eds. , Plenum Press, Ne
w York, 1995, pp 821-902).

The immunostimulatory activity of a sperm surface protein antigen can be determined by monitoring the immune response in a test animal after immunization with a sperm surface protein antigen, or by using any immunoassay known in the art. . Generation of a humoral (antibody) response and / or generation of a cell-mediated noodle preparation can be employed as an indicator of an immune response. Test animals can include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and can ultimately be human subjects.

The method of introducing the vaccine can be oral, intravaginal, intradermal,
Through intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and incision (scratching through the upper layers of the skin (eg, using a bifurcated needle))
Or any other standard route of immunization. The immune response of a test subject can be analyzed by a variety of approaches, such as: the responsiveness of the resulting immune serum to egg protein antigen, which is assayed by the following known techniques: Assay (ELISA), immunoblot, radioimmunoprecipitation, etc., or if the egg protein antigen exhibits antigenicity or immunogenicity, by protecting the immunized host against fertility in the immunized host).

As an example of a suitable animal test for a sperm surface protein vaccine, the vaccine of the present invention can be tested in rabbits for the ability to elicit an antibody response to a sperm surface protein antigen. Male specific pathogen skin containing (SPF) young adult New Z
eland White rabbits can be used. Each of the test groups receives a fixed concentration of vaccine. The control group receives an injection of 1 mM Tris-HCl pH 9.0 without sperm surface protein antigen.

Blood samples can be taken from the rabbit every one or two weeks and serum can be analyzed for antibodies to sperm surface proteins. The presence of an antibody specific for this antigen can be assayed using, for example, an ELISA. Effective doses (immune doses) of the vaccines of the invention can be extrapolated from dose response curves derived from animal model test systems.

Suitable preparations of such vaccines include: injectables (either as liquid solutions or suspensions); suitable for liquids for injection, suspensions therein or liquids prior thereto. Solid forms can also be prepared. The preparation can also be emulsified, or the polypeptide can be encapsulated in liposomes. Active immunogenic components are often mixed with excipients. The excipient is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like and combinations thereof. In addition, if desired, the vaccine preparation can also contain trace amounts of auxiliary substances (eg,
A wetting or emulsifying agent, a pH buffering agent, and / or an adjuvant,
(Enhancing the efficacy of the vaccine).

Examples of adjuvants that may be effective include, but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D
-Isoglutamine (thr, MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl -D-isoglutaminyl-L
-Alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine, as well as surface-activating substances such as lysolecithin, pluronic polyols; polyanions; peptides; oil emulsions; And MDP. The potency of the adjuvant can be determined by measuring the induction of antibodies directed against the immunogenic polypeptide comprising the egg surface protein polypeptide epitope. The antibody results from administration of the polypeptide in a vaccine. This vaccine is also composed of various adjuvants.

[0046] The immunogen may comprise one or more polypeptides comprising the amino acid sequences of SEQ ID NOs: 1-23. In addition, the immunogen can also be incorporated into liposomes, or conjugated to polysaccharides and / or other polymers for use in vaccine formulations. If the recombinant antigen is a hapten (ie, a molecule that is antigenic in that it can selectively react with cognate antibodies, but is not immunogenic in that it cannot elicit an immune response), A hapten can be covalently linked to a carrier or can be an immunogenic molecule;
Large proteins, such as serum albumin, confer immunogenicity to the hapten bound thereto. Hapten carriers can be formulated for use as vaccines.

The polypeptide may be formulated into neutral or salt serum. Pharmaceutically acceptable salts include: acid addition salts (formed with the advantageous amino groups of the peptide), and inorganic acids (eg, hydrochloric or phosphoric acid) or organic acids (
Acid addition salts formed with, for example, acetic acid, oxalic acid, tartaric acid, malic acid and the like). Salts formed with free carboxy groups also include inorganic bases (eg, sodium, ammonium, calcium, or iron (III) chloride) and organic bases (eg, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine) , Procaine, etc.).

[0048] The vaccine of the present invention may be multivalent or monovalent. Multivalent vaccines are made from recombinant viruses that direct the expression of more than one antigen. The patient to which the vaccine is administered is preferably a mammal, and preferably a human, but can also be a non-human animal, including but not limited to: cows, horses,
Sheep, pigs, birds (eg, chickens), goats, cats, dogs, hamsters, mice and rats.

The vaccine formulation of the invention comprises an effective immunizing amount of a sperm surface protein and a pharmaceutically acceptable carrier. A vaccine preparation comprises an effective immunizing amount of one or more antigens and a pharmaceutically acceptable carrier. The carrier is preferably sterile. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The formulation should suit the mode of administration.

Generally, the components are mixed separately or together in unit dosage form (eg, a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of active agent). Supplied either. When the composition is administered by injection, a sterile diluted ampoule may be provided so that the contents may be mixed prior to administration. The invention also relates to one of the vaccine formulations of the invention.
There is provided a pharmaceutical pack or kit comprising one or more containers containing one or more components.

One aspect of the present invention pertains to antibodies (monoclonal or polyclonal) that bind to SSP. In particular, those antibodies that immunospecifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23. In one preferred embodiment, the antibody is a monoclonal antibody. Antibodies generated against the SSP antigens of the present invention have potential use in vaccination against insemination, infertility, diagnostic immunoassays, passive immunoassays and the generation of anti-idiotypic antibodies. Accordingly, the present invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an anti-sperm surface protein antibody in an amount sufficient to inhibit insemination. In an immunization procedure, the amount of immunization used and the immunization schedule are determined by one of skill in the art and are administered by taking into account the subject's immune response and antibody titers.

According to one embodiment, a method is provided for preparing a contraceptive antiserum. Here, this antiserum contains an antibody against the sperm surface antigen. In a preferred embodiment, the sperm surface antigen is a protein specific to male gametes, more specifically sperm. The method comprises immunizing an animal with a sperm surface protein or an immunogenic fragment thereof, obtaining a serum sample from the immunized animal, and determining the ability to bind to the serum surface protein (or immunogenic fragment thereof). Screening the serum and collecting the serum based on its ability to bind to sperm surface proteins. The resulting serum may contain polyclonal antibodies, or may contain one or more monoclonal antibodies. Alternatively, antibody screening cells can be obtained from the immunized animal, and the cells can be immortalized and screened for cells secreting antibodies that bind to sperm surface proteins using standard techniques. . Cells secreting antibodies that bind to sperm surface proteins can be isolated and used to produce antibodies.

The antibodies generated can be isolated by standard techniques known in the art (eg, immunoaffinity chromatography, centrifugation, sedimentation, etc.) and used in diagnostic immunoassays. These antibodies can be used to monitor treatment and / or disease progression. Immunoassay systems known in the art can be used for this purpose, including but not limited to: competitive and non-competitive assays using the following techniques: radioimmunoassay,
ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassay, precipitin reaction, gel nucleic acid precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay And immunoelectrophoresis assays.

[0054] The vaccine formulations of the present invention can also be used to raise antibodies for use in passive immunotherapy. Here, short-term protection of the host can be achieved by administration of a preformed antibody that is considered against a heterologous organism.

[0055] Antibodies produced by the vaccine formulations of the present invention can also be used in the production of anti-idiotype antibodies. This anti-itiotype antibody can then be used for immunization to produce an antibody subpopulation that binds to the primary antigen of the pathogenic microorganism (Jerne, 1974, Ann Immunol. (Paris) 125c).
: 373; Jerne, et al., 1982, EMBO J .; 1: 234).

Various delivery systems are known and can be used to administer the therapeutics of the invention (eg, liposomes, microparticles, encapsulation in microcapsules, sets capable of expressing the therapeutics). Recombinant cells, constructs of a therapeutic nucleic acid as part of a retrovirus or other vector). Methods of introduction include, but are not limited to:
Intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound can be administered by any convenient route, eg, by injection or by bolus injection, by absorption through an epithelial lining or mucosal lining (eg, oral, rectal and intestinal mucosa, etc.), and by other routes. Can be administered with a chemically active agent. Administration can be systemic or local. Pulmonary administration can also be employed by the use of inhalers or nebulizers, and formulations with aerosolizing agents. In certain embodiments, the pharmaceutical compositions of the invention are administered locally to the area in need of treatment It may be desired. This may be achieved, for example and without limitation, by topical application, injection, means of a catheter, means of a suppository, or means of an implant. This implant is a porous, non-porous or gelatinous material,
This includes a membrane (eg, a sialastic membrane) or a fiber.

In another embodiment, the therapeutic agent can be delivered in a vesicle, especially a liposome (see: Langer, Science 249: 1527).
-1533 (1990); Treat et al., Liposomes in the.
Therapy of Infectious Disease and Ca
ncer, Lopez-Berstein and Fidler (eds.), L
iss, New York, pp. 353-365 (1989); Lopez-.
Berstein, ibid. 317-327; see generally ibid.).

In yet another embodiment, the therapeutic agent can be delivered in a controlled release system. In one embodiment, a pump may be used (see: La
nger, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14: 201 (1987); Buchwald et al., Surgery 8.
8: 507 (1980); Saudek et al. Engl. J. Med. 321
: 574 (1989)). In another embodiment, a polymerizable material may be used (
See: Medical Applications of Con.
trolled Release, Langer and Wise (ed.), C
RC Pres. , Boca Raton, Florida (1974); Co.
controlled Drug Bioavailability, Drug P
product Design and Performance, Smolen
And Ball (eds), Wiley, New York (1984); Rang.
er and Peppas, J. et al. Macromol. Sci. Rev .. Ma
cromol. Chem. 23:61 (1983); see also: L
evy et al., Science 228: 190 (1985); During et al.,
Ann. Neurol. 25: 351 (1989); Howard et al. Ne
urosurg. 71: 105 (1989)). In yet another embodiment,
A controlled release system can be placed near the therapeutic target, ie, in the testes. Therefore,
Only a portion of the systemic volume is required (see, eg, Goodso
n, 1984, in Medical Applications of Co.
controlled Release, supra, vol. 2, pp. 115-13
8).

The present invention also provides a pharmaceutical composition. The pharmaceutical compositions of the invention include a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier. In a preferred embodiment, the composition is formulated in accordance with conventional means as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition also includes a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the components are supplied separately or mixed together in unit dosage form (for example, a dry lyophilized powder or anhydrous concentrate in a sealed enclosure such as an ampoule or sachet, indicating the amount of active agent). . When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.

[0060] The amount of a therapeutic agent of the invention that is effective in a particular treatment or condition will depend on the nature of the disorder or condition. And it can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. Precise dosages to be used in the formulation will depend on the route of administration, and should be determined according to the judgment of the practitioner and each patient's circumstances. However, a suitable dosage range for intravenous administration is generally 1
20-500 μg of active ingredient per kg body weight. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10%
Contains ~ 95% active ingredient. A suitable dosage range for intranasal administration is about 0.01
pg / kg body weight to 1 mg / kg body weight. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Example 1 Materials Human Serum Serum was obtained from infertile men and women with “unexplained” infertility. Infertile subjects did not have any previously diagnosed hormonal, infectious, or physical cause for their infertility. The male subject had not undergone vasectomy.

Immune Bead Binding Anti-sperm antibodies in the sera of infertile female patients were detected by indirect immunobead binding, as described below: Manual of clinical
laboratory immunology, 4th edition, Americans
ociety for microbiology, Washington, D
. C 1992; 1013-1017. Anti-sperm antibodies bound to sperm of male patients were detected by direct immunobead binding. All serum and serum samples
Tested for the presence of anti-sperm antibodies specific for IgG, IgM and IgA, and 18 infertile male subjects, 9 infertile female subjects, 5 fertile male subjects and 5 fertile female subjects Body included.

Criteria for Selecting Serum for Western Blot Analysis A high IBT score (ie, greater than 60% of sperm binding to the beads was observed, indicating that IgG and / or IgM specific Sera for further studies were selected based on the anti-sperm antibody index). All sera selected contained antibodies directed against the sperm head or whole sperm. A total of 15 sperm samples were selected for infertile men for Western blot analysis and six serum samples were selected from infertile women.

Preparation of Sperm Antigen Semen specimens were obtained from a fertile donor with normal sperm quality. Only ejaculates with normal semen properties were used in this study. After semen liquefaction, mature sperm
semicollar, immature germline cells and non-sperm cells (mainly leukocytes and epithelial cells) from Percoll (Pharncia Biote)
ch, Uppsala, Sweden) density centrifugation (described by:
Naaby-Hansen et al. (Biol Reprod 1997; 56:77).
1-787)). Before the last centrifugation, count the cells,
And all samples showed greater than 90% motility. Sperm from 8-12 individuals were pooled and frozen immediately until further use. All samples were submitted to the University of Virginia Human I
Obtained under informed consent using a format approved by the Nvestigation Committee.

Radioiodination Radioiodination was performed as described by Naby-Hansen et al., (Biol Reprod.
1997; 56: 771-787) and performed according to standardized procedures. The sperm purified by percoll was subjected to 20 ×
To a final concentration of 10 ⁶ cells / ml, it was suspended in Ham's F-10 medium. Washed iodine beads (Pierce Chemical Co., Rockford)
, IL) (one bead per 8 × 10 ⁶ sperm) and ¹²⁵ I-Na without carrier (10 μCi / 10 ⁶ sperm) were added to the sample. Radiolabeling was performed by incubating the samples on a shaking table at 20 ° C. for 10 minutes. Cells were removed from iodine-beads by pipetting and immediately subjected to a second round of Percoll density gradient centrifugation. Thereafter, the cells were washed three times in Ham's F-10 medium. The "surface" proteins studied here are primarily plasma membrane proteins that are accessible to external labels on ejaculated sperm. These consist of almost intact cells that do not undergo an acrosome reaction, according to the preparation method used. Cells are counted before the last wash,
The resulting pellet was used for sperm protein extraction. Autoradiography was performed using a sandwich of components in the following order: intensifying screen, blot, two layers of film, and intensifying screen. X-ray films were usually exposed for 3 weeks.

(Solubilization Procedure) Spermatozoa were treated with 100 mM dithiothreitol (DTT), urea (9.8 M), and protease inhibitor (2 mM PMSF, 5 mM iodoacetamide, 5 mM EDTA, 3 mg / ml L- 1-chloro-3- (4-tosylamido) -7-amino-2-heptanone-hydrochloride (TLCK), 1.4
2 containing 6 mM pepstatin A and 2.1 mM leupeptin)
% Octyl-β-gluco-pyranoside (OBG) was solubilized in lysis buffer. 5 × 10 ⁸ cells / ml were solubilized by constantly shaking at 4 ° C. for 45 minutes. Insoluble material was removed by centrifugation at 10,000 xg for 2 minutes, and the supernatant was subjected to one-dimensional electrophoresis.

Electrophoresis Isoelectric focusing was performed using gel compositions (Naaby-H) as described previously.
Ansen et al., Biol Reprod 1997; 56: 771-787). The carrier ampholine composition comprises 20%
PH 5-7, 20% pH 7-9, and 60% pH 3.5-10. 65 μl of sperm extract (approximately 0.15 mg of protein) was applied per rod. Tubes were loaded with 5% NP-40, 1% ampholine (pH 3.5).
-10), the sample was filled by gentle overlaying with a buffer containing 8 M urea and 100 mM DTT. Isoelectric focusing was performed at a total of 17,700 volts-h using stepped voltages: 2 hours at 200 v, 5 hours at 500 v, 11 hours at 800 v, and 3 hours at 2000 volts.

Non-equilibrium pH gradient electrophoresis (NEPHGE) was performed as described in Celis et al., Electro.
phoresis, 1992; 13: 893-959) and Naaby-Ha.
Performed in 14 x 0.15 cm acrylamide rods using the gel composition described by Nsen et al. (Biol Reprod 1997; 56: 771-787). The carrier ampholine composition is 13% pH 3.5-10
37% pH 5-8, 13% pH 6.5-9, and 37% pH 8-10
. It was 5. 55 μl (approximately 0.13 mg of protein) of sperm extract was applied per rod. Electrophoresis was performed at a total of 8600 volt-h using 5 hours at 400 volts and 3 hours at 550 volts.

Two-dimensional SDS-PAGE was performed in a 0.15 cm thick, 16 × 16 cm slab gel using a Protean II xi Multi-cell apparatus (Bio-
Rad, Richmond, CA) (Biol Reprod 1997; 56).
: 771-787) using a linear gradient gel (T = 9-15%;
T = concentration of acrylamide in gel). Electrotransfer to the nitrocellulose membrane was performed as described in Biol Reprod 1997; 56: 771-787.

(Immunoblotting Analysis) After the electrotransfer of the polypeptide separated by 2-D, the membrane was washed with PBS (pH 7.4).
), Rinsed twice for 5 minutes and the excess binding sites were removed at room temperature in PBS containing 5% milk powder and 0.05% Tween 20 at room temperature.
Blocked by incubation for hours. Blots were incubated with serum at a 1: 2000 dilution overnight at 4 ° C. with constant gentle shaking. In some cases, a given blot was incubated with one or more sera. In these examples, blots were washed in PBS containing 0.05% Tween 20 (pH 7.4) after overnight incubation at 4 ° C. with the first serum, and the same as above. Incubated with another serum under conditions. Fifteen sera from infertile men were analyzed and six sera from infertile women were analyzed. These included anti-sperm antibodies as shown by IBT. Secondary enzyme-linked antibody (Jackson Immu
noResearch Lab. , WestGrove, PA) (goat anti-human I)
gG + IgM) in PBS containing 0.05% Tween 20:
Diluted to 5000 and incubated blot at 20 ° C. for 1 hour. The secondary antibody alone did not bind to the blotted human sperm proteins (data not shown). Horseradish peroxidase conjugate was visualized by enhanced chemiluminescence (ECL) using the manufacturer's protocol (Amersham Corp., UK).

Scan and Computer Analysis X-ray films from chemiluminescent preparations and autoradiograms from radioiodination experiments were taken with a Kodak camera (Eastman Kodak, Roches).
ter, NY). The obtained 2-D image was converted to Bio Ima
Ge "2D analyzer" version 6.1 (Bio Image, Ann Arb)
or, MI). The software automatically identifies, quantifies, and compares protein spots separated by 2D gels. By using a composite image showing spots of aggregated proteins recognized by fertile subjects of both genders, using a protein spot that is common to each image as a reference point or `` anchor '', Computer-scanned images of ECL blots corresponding to male and female individuals were obtained after fitting. Anchors were selected based on electrophoretic mobility, locus of protein spots, and spot shape. Compare the resulting composite images of the separated protein spots to subtract the protein spot recognized by the fertile subject from the protein spot recognized by the infertile subject, and And matched images corresponding to groups of women. In this way, major autoantigens and isoantigens that are uniquely recognized by infertile subjects are identified.

A database of sperm surface proteins (encyclopedia of sperm surface) was created according to the fit of the autoradiogram obtained after radioiodination of the surface. Care was taken to include only those spots that were consistently labeled.
Computer generated images containing autoantigens and isoantigens were matched to an encyclopedia of the sperm surface to identify surface autoantigens and isoantigens.

Results All the infertile sera tested (15 men and 6 women) contained IgG and / or IgM anti-sperm antibodies and more than 60% of sperm bound to beads in IBT did. The control sera used from fertile individuals (5 men and 5 women) were negative for IBT, with less than 10% of sperm bound by the immunobeads.

Silver Staining of Human Sperm Protein Solubilized with Nonionic Detergent / Urea 2
-D-gel (16 x 15 cm), IEF / PAGE and NEPHGE /
More than 1300 protein spots by PAGE were determined. These gels show a repertoire of protein spots from a sperm pool of 10 individuals. Although the protein pattern is highly reproducible, a small variation between donors was observed with respect to relative abundance and charge of the same protein (these vary when individual samples are compared by computer analysis). Found to work). The identity of some of the major protein spots was identified by microsequencing individual spots or by immunoblotting with antisera against specific proteins. For example, members of a family of different heat shock proteins were determined. These proteins are abundant on sperm membranes. PH-20 (sperm hyaluronidase) is a membrane-bound protein and is presented by three 53 kDa isoforms. The major cytoplasmic proteins, α-tubulin and β-tubulin, were also detected as being human human sperm calreticulin.

[0075] 2D Western blots of sperm proteins determined by IEF or NEPHGE were probed with serum from fertile male subjects or with serum from infertile male subjects. Sera from infertile subjects showed stronger immunoreactivity to some spots than fertile subjects,
The significance of such differences in intensity is not currently understood. In addition, a number of antigenic spots in both acidic and basic proteins were reactive with infertile sera, but not with fertile sera. Similarly, differences in immunological reactivity were noted by comparing Western blots probed with infertile female sera to those that were reactive with fertile female sera; serum of infertile subjects was noted. Recognizes more antigenic spots, and also
It showed stronger reactivity to some spots than sera from fertile women.

Analysis of Acidic Antigens Analysis of individual infertile sera showed significant variability in the range of recognized specific proteins, although some antigens may be common for different individuals. Also demonstrated. This observation of the range of recognized sperm antigens implies considerable diversity in the repertoire of anti-sperm antibodies in human males. On the other hand, we often focused on similarities in patterns of immunoreactivity for specific sperm antigens. By computer analysis and comparison of blots reacted with infertile and fertile sera, a spot of 4 proteins at approximately 60 kDa was identified by both infertile individuals belonging to a subset of proteins not recognized by fertile subjects. It was shown that it was recognized.

A blot of the repertoire of immunoreactive sperm proteins recognized by fertile and infertile sera from both genders, together with five sera from each group of subjects, was Obtained by successive incubations (FIGS. 1A to 1D). Sera from infertile subjects were selected based on their high immunoreactivity, heterogeneity in immunoreactivity, and unique recognition of specific protein spots after individual Western blot analysis of the sera. 1A and 1B show a repertoire of sperm proteins bound by serum from five fertile male (A) and five fertile female (B) subjects. Comparison of sperm antigens recognized by fertile male (A) and fertile female (B) subjects revealed significant overall similarity in the repertoire between the two genders, Fertile male sera recognized more antigens than fertile female sera. 1C and 1D show Western blots sequentially probed with sera from five infertile men (C) and five infertile female subjects (D), respectively. Comparison of Western blots probed with sera from fertile patients and sera from infertile patients (A + B vs. C + D in FIG. 1) shows that several different sperm antigens were recognized by the infertile patients (Arrow). The molecular weight of the prominent protein spot uniquely recognized by male patients is 34 kDa
(PI 4.2), 42 kDa (pI 4.3), 88.5 kDa (pi 4.0).
0), 106 kDa (pI 4.2), 41.8 kDa (pI 5.3, pI
5.4, pI 5.5), 61.5 kDa (pI 5.62), 60.4 kDa
(PI 5.75), and 60 kDa (pI 6.6 and 6.7). The major isoantigen recognized by infertile women is 60 kDa (pI 6.2,
pI 6.4, pI 5.82, pI 5.88), 32.3 kDa (pI 6
. 7, pI 6.8, pI 6.85), and 41.2 kDa (pI 5.1
).

Changes in immunoreactivity between sera of male and female patients. Computer image analysis of immunoblot showed that, depending on the sera of infertile male patients, a total of 310 antigens were recognized. On the other hand, it was shown that 205 antigens were recognized by the serum of infertile female patients. This result suggests that the immunoreactivity to sperm antigens exhibited by infertile men is probably higher than that of infertile women. In addition, different changes in the pattern of immunoreactivity are distinguishable between the two genders, each of these groups showing specificity for a different set of proteins (FIGS. 1C and 1D), although both subjects Proteins that were common to body groups were noted.

Computer-assisted subtraction of the antigens recognized by both male and female fertile subjects from the entire repertoire of antigens recognized by the group of infertile men yielded 86 protein antigens. It became clear. These were recognized only by infertile male subjects (FIG. 2A) as compared to fertile subjects. Of these 86 antigens recognized only by infertile male patients, 72 were specific only to the serum of infertile male patients, and they were not recognized by the serum of infertile women. A similar computer-assisted analysis of the 2-D blot yielded 26 protein spots that were unique for infertile female subjects (FIG. 2B). Twelve of these antigens were specific only to infertile female patients and were not recognized by infertile male patients. Among the sperm antigens recognized only by the serum (both male and female) of the infertile patient,
Four antigens were commonly recognized by the sera of both male and female patients (arrows).

Sperm Surface Antigen Recognized Only by ASA Positive Patients FIG. 3 shows the results obtained after computer scanning of autoradiograms obtained by 2D IEF-PAGE of vectorly iodinated sperm proteins. Also, a computer generated composite image of human sperm surface protein is shown (Biol Reprod 1997; 56:
771-787). 103 proteins were identified on the surface of sperm by this method. Computer-aided adaptation of sperm autoantigens and isoantigens using composite images of sperm surface proteins provided identification of a subset of sperm surface autoantigens and isoantigens. The positions of the autoantigen and isoantigen on the surface of the six spermatozoa are shown in FIG. 3 (arrows). Notably, the pI of 6.2 and 6.4
Are two 60 kDa proteins that were recognized by the sera of two infertile women. Two of the sperm antigens recognized by infertile male sera recognized an 88.2 kDa sperm surface protein with a pI of 4.0. The two sera were also 34 kDa and 38 kDa, both with a pI of 4.2.
Recognized two sperm surface proteins with molecular weights of Table 1 lists the mass and pI of iso- and autoantigens on the surface of sperm.

[0081]

[Table 1] Correlation between Western Blot Results and IBT Results These sera that yielded high scores in the immunobead test for binding to various surface domains were used to recognize sperm surface proteins on Western blots. Note that we included the antibodies shown in the 2D analysis,
It was interesting. Table 1 also describes the immunobead patterns of the sera of these subjects. Each of these sera showed IBT reactivity to IgG and / or IgM specific antibodies. Antibodies were directed against the head or whole surface of the sperm.

Discussion Specific sperm proteins were recognized and identified on 2-D gels by sera from anti-sperm antibody-positive infertile subjects. These antigens probably play a significant role in producing immune infertility, and may play a role in the events of the fertilization cascade. Many studies performed in the past using western blotting of one-dimensional gels have yielded varying results with respect to the molecular weight of sperm antigens recognized by sperm containing anti-sperm antibodies. Most of these studies were performed on infertile individuals, and the response of sperm antigen using fertility control sera was often not evaluated. In addition, conflicting reports on the molecular weight of reactive sperm antigens may be due to changes in sperm extraction procedures. In the present invention, two-dimensional electrophoresis was used for separating proteins based on the isoelectric point of individual proteins in one dimension, and for separating proteins based on molecular weight in two dimensions. This may provide better analysis of the protein, and may serve as a prelude to microsequencing.

The high resolution 2-D gel technology, which is optimal for separating sperm proteins, is suitable for screening serum for anti-sperm antibodies. Initial screening of sera from infertile patients for the presence of sperm surface antibodies by IBT provided additional focus by turning attention to sera containing anti-sperm antibodies. These are associated with antibody-mediated events of aggregation, cytotoxicity, or blocking at the sperm surface, and thus are functionally associated with infertility.

[0084] Immunoblot analysis of individual sera implies that there is considerable diversity in the repertoire of anti-sperm immunoglobulins. The finding in this study that many patients did not recognize the same set of antigens may be due in part to the complexity of sperm assembly and the diversity of antigens involved in the autoimmune response. It is also likely that the mechanisms and patterns of infertility in these patients may be different.
Antibodies that recognize different sperm surface proteins can affect sperm's ability to fertilize by acting at different stages of the infertility process. Differences in the repertoire of antibodies can also be related to differences in the mode of immunization. Previous studies have shown that the subclass of anti-sperm IgA antibodies is altered in humans with autoimmunity to sperm (Bronson et al., Human sperma).
nantibodies and their detection: Manual
of clinical laboratory immunology, 4th edition, American society for microbiology
, Washington, D.C. C. 1992; 1013-1017).

The Western blot data (see FIGS. 1A and 1B) confirm the presence of sperm autoantibodies in a high percentage of both fertile and infertile individuals, while fertile individuals Recognized much less sperm antigen, and the relative reactivity intensity was weaker compared to infertile individuals. These data imply that most of the sperm antigens recognized by fertile sera are endogenous, as each of these sera was negative for anti-sperm antibodies in the immunobead test. Some of these intracellular antigens have been microsequenced and identified as components of sperm-specific structures such as the fibrous sheath and outer dense fibers. In the present invention, the systematic identification of a subset of these antigens results in the production of sperm antigens, which trigger the production of functionally related and / or irrelevant antibodies, which are (Which may have a role).

[0086] Continuous incubation of sera for immunoblotting in combination with computer analysis has been successful in providing infertile patients with information about unique major autoantigens and isoantigens. Male sera recognized more antigen than female sera (310 vs. 205), but extensive evaluation using samples from a higher number of female subjects makes the results statistically valid In order to be needed. Longer constant exposure of sperm proteins to the immune system in male patients may explain higher reactivity in males. Furthermore, clear differences in the pattern of immunoreactivity between male and female patients were identifiable. The serum of male patients is reactive to some acidic proteins (p
I 4.0 to 5.0), which were not recognized by any of the analyzed sera from women (see FIGS. 1C and 1D). Conversely, major isoantigens recognized by female proteins in the pi range of 6.0 to 7.5 were less reactive to serum from male patients (FIGS. 1C and 1D).

[0087] A reasonable criterion for identifying fertility associated with sperm antigens is by the identification of those antigens that are present on the surface of cells. It is therefore particularly important to distinguish between immunity to sperm surface antigens and immunity to sperm internal antigens. For this reason, immobilized sperm sperm surface protein, which has a damaged membrane,
Rather than labeling (which allows labeling of internal proteins), the surface proteins of living sperm were labeled. The surface proteins of sperm, ¹²⁵ I
Followed by IEF / PAGE and NEPHGE / PAGE separations (Naaby-Hansen et al., Biol Reprod 1997;
56: 771-787). Using computer analysis, IEF
A composite image consisting of 103 surface labeled proteins in a / PAGE gel was obtained. This computer image may play a role in immune infertility.
It was possible to classify a subset of individual sperm surface antigens. One of these antigens was recognized by two male infertile patients at a pI of 4.0 and 8
It is a 8.2 kDa protein. Naby-Hansen et al. (Biol R
eprod 1997; 56: 771-787) has previously shown this protein to be a sperm surface protein phosphorylated at tyrosine residues. Interestingly, the sera of the two women showed significant similarities in immunoreactivity, which recognized the same protein spot of 60 kDa at pi of 6.2 and 6.4. Both of these sperm proteins were found to be present on the surface of the sperm, as evidenced by the labeling of the sperm surface with radioactive ^125I . In addition, both sera contained IgG antibodies directed against the sperm head, as shown by the immunobead test. This allows these antigens to
It was confirmed that it was exposed on the surface of the spermatozoa, possibly located on the sperm head. The sera of two male patients strongly recognized two protein spots of 34 kDa and 38 kDa identified on the surface of sperm.

Current analysis of serum based on both IBT and Western blotting to diagnose immunity to sperm can detect the presence of anti-sperm antibodies and identify alloantigens on the surface of sperm However, the identification of the epitopes directed by these antibodies should improve the reliability of this analysis. Recognition of multiple antigens that appear to have potential roles in fertility can occur due to a common epitope.

Previous reports have shown that monoclonal antibodies directed against different antigens of the same isoform, but present on the sperm head, have been shown to produce zona pellucida or sperm-egg.
Has been shown to be able to block sperm adhesion to the fusant. In addition, antibodies directed against different epitopes of the same sperm surface antigen may also affect sperm fertility in different ways.

The computerized digitization of information about the 2-D gel pattern of surface-labeled sperm proteins opens up new tools in the diagnosis of anti-sperm antibodies, and the potential for treatment of antibody-mediated infertility. Sperm proteome (prot)
eome) and based on indicators of sperm surface proteins, 2-
D immunoblotting is used herein to identify such patients with antibodies to protein antigens that are accessible on the cell surface, thereby obtaining more specific information about the patient's repertoire of autoantibodies or isoantibodies. Can be used for These databases are expanded with the identification of additional autoantigens or isoantigens, and defined natural or recombinant as fine sequence information and fine sequences based on cloning provide relevant protein characteristics. The potential for rational criteria for antibody-mediated infertility immunotherapy using sperm antigens of the present invention may become apparent. In addition, diagnosis of antibody-mediated infertility can be simplified using 2-D immunoblots in patient sera and by referencing the results for a database of surface antigens.

Example 2 Sequence analysis of two 2D gel bands. Identification of Hyaluronic Acid Binding Protein (Peptide No. 1, Table 1) (Method :) The upper and lower bands migrated at approximately 36 kDa with a pI of 4.4. The band was cut from the gel in close proximity to minimize excess polyacrylamide as much as possible, divided into a number of smaller fragments, washed, and 500 μL of 5
Decolorized overnight in 0% methanol. Dehydrate the gel fragments in acetonitrile,
Rehydrated in 50 μL of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at 55 ° C. for 1 hour. The DTT solution was removed and the sample was alkylated in 50 μL of 50 mM iodoacetamide / 0.1 M ammonium bicarbonate for 1 hour at room temperature in the dark. The reagent was removed and the gel fragment was washed with 100 μL of 0.1 M ammonium bicarbonate and 100 μL
In acetonitrile for 5 minutes. Acetonitrile was removed and gel fragments were rehydrated in 100 μL of 0.1 M ammonium bicarbonate. Fragment
Rehydrated in 100 μL of acetonitrile to remove acetonitrile, and the fragments were completely dried by vacuum centrifugation. Gel fragments were rehydrated in 12.5 ng / μL trypsin in 50 mM ammonium bicarbonate and incubated on ice for 45 minutes. All excess trypsin solution was removed and 20 μL of 50 mM ammonium bicarbonate was added. The sample was digested overnight at 37 ° C. and the peptide formed was extracted from two 200 μL of polyacrylamide in 50% acetonitrile / 5% formic acid. These extracts were combined and evaporated to <20 μL for LC-MS analysis.

The LC-MS system is a 10 cm × 75 μm id POROS 10 RC
Finn with an electrospray ion source connected to a reversed-phase capillary column
igan-MAT TSQ7000 system. A volume of 1 μL of the extract was injected, and the peptide was eluted from the column with an acetonitrile / 0.1% acetic acid gradient at a flow rate of 0.6 μL / min. Electrospray ion source is set to 7
1.2 μL / min coaxial sheath liquid flow of 0% methanol / 30% water / 0.125% acetic acid, adjusted as needed for optimal sensitivity A coaxial nitrogen stream (nitrogen f)
low) and operated at 4.5 kV. The digest was analyzed by capillary LC electrospray mass spectrometry to determine the molecular weight of the peptide present in the digest. The peptide sequence for the detected peptide was determined by collisionally activated dissociation using LC-electrospray tandem mass spectrometry using argon as collision gas.

Results: The molecular weights of these two digests determined by LC-MS analysis and the amino acid sequences of the peptides determined by LC-tandem MS are shown in Table 2. 6 peptides were stained darker band LB3-83-2 (Anne, bottom)
And four of these same peptides (peptides # 2, 3, 4,
And 5) were detected in the brighter stained band LB-3-83-1 (Anne, top). Sequence information was obtained for five of the six peptides. Database searches using CAD spectral information (SEQUEST) and database searches using partial peptide sequences (BLAST)
Two peptides in the database sequence of the human hyaluronic acid binding protein were identified and numbered 3 and 4 (NCBInr. 6.8.97 accession number 730).
772, calculated MW = 31.4 kDa, calculated pI = 4.3). No other peptides were found in this or any other database sequence.

Taken together, both bands give rise to a similar series of peptides. Both bands contain two peptides, which match the database sequence of human hyaluronic acid binding protein. Both bands contain additional peptides, which are
It cannot be matched against peptides in any database, so they appear to be from a second unknown protein.

[0095]

[Table 2] ¹ X represents I or L. These cannot be distinguished by low energy CAD. Mo indicates oxidized M. Lowercase letters indicate experimental assignments. _
Indicates a single unknown amino acid, ---- indicates an unknown number of unknown amino acids. The abbreviations are as follows: “X” = Ile or Leu; SVXXSXK indicates the following amino acid sequence: Ser Val Ile Leu Ser Leu Lys (SEQ ID NO: 24), Ser Val Leu Ile Ser Leu Lys (SEQ ID NO: 25), Ser Val Leu Leu Ser Ile Lys (SEQ ID NO: 26), Ser Val Ile Ile Ser Leu Lys (SEQ ID NO: 27), Ser Val Ile Ile Ser Ile Lys (SEQ ID NO: 28), SerVal SEQ ID NO: 29),
And Ser Val Leu Leu Ser Leu Lys (SEQ ID NO: 30); VYTS shows the following amino acid sequence: Val Tyr Thr Ser Ser (SEQ ID NO: 31); GGWEXNGNGTEAK shows the following amino acid sequence; Gly Gly Ile Asn Gly
Thr Glu Ala Lys (SEQ ID NO: 32), Gly Gly Trp Glu Ile Glu Leu Asn Gly
Thr Glu Ala Lys (SEQ ID NO: 33), Gly Gly Trp Glu Leu Glu Ile Asn Gly
Thr Glu Ala Lys (SEQ ID NO: 34), and Gly Gly Trp Glu Leu Glu Leu Asn Gly
AFGDFXSDEXKEER has the following amino acid sequence: Ala Phe Val Asp Phe Ile Ser Asp Glu; Thr Glu Ala Lys (SEQ ID NO: 35);
Ile Lys Glu Glu Arg (SEQ ID NO: 36), Ala Phe Val Asp Phe Ile Ser Asp Glu
Leu Lys Glu Glu Arg (SEQ ID NO: 37), Ala Phe Val Asp Phe Leu Ser Asp Glu
Ile Lys Glu Glu Arg (SEQ ID NO: 38), and Ala Phe Val Asp Phe Leu Ser Asp Glu
Leu Lys Glu Glu Arg (SEQ ID NO: 39); and SGGWELELNGTEAK show the following amino acid sequence: Ser Gly Gly Trp Glu Leu Glu Leu Asn
Gly Thr Glu Ala Lys (SEQ ID NO: 40).

Example 3 Sequence analysis of three CM-stained 2D gel bands (Peptide No. 2, Table 1). Method: The gel fragments were transferred to a siliconized tube, washed and washed.
Decolorized overnight in 0 μL of 50% methanol. Gel fragments were dehydrated in acetonitrile, rehydrated in 30 μL of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate, and reduced for 0.5 hours at room temperature. The DTT solution was removed and the sample was washed with 30 μL of 50 mM in 0.1 M ammonium bicarbonate.
In iodoacetamide for 0.5 h at room temperature. Remove the reagents,
The gel fragments were then dehydrated in 100 μL of acetonitrile. Acetonitrile was removed and gel fragments were rehydrated in 100 μL of 0.1 M ammonium bicarbonate. Fragments were dehydrated in 100 μL of acetonitrile to remove acetonitrile, and the fragments were completely dried by vacuum centrifugation. Gel fragments were rehydrated in 20 ng / μL trypsin in 50 mM ammonium bicarbonate for 10 minutes on ice. Remove any excess trypsin solution and add 20 μl
L of 50 mM ammonium bicarbonate was added. The sample was digested overnight at 37 ° C. and the peptide formed was combined with two 30 μL of 50% acetonitrile / 5%
Extracted from polyacrylamide in an aliquot of formic acid. These extracts were combined and evaporated to 25 μL for LC-MS analysis.

The LC-MS system was self-packed 8 cm ×
The system consisted of a Finnigan LCQ ion trap mass spectrometry system equipped with a Protana nanospray ion source connected to a 75 μm id Phenomenex Jupiter 10 μm C18 reversed phase capillary column. A volume of 0.5-5 μL of the extract is injected and the peptide is
The column was eluted with an acetonitrile / 0.1% acetic acid gradient at a flow rate of 0.25 μL / min. The nanospray ion source was operated at 2.8 kV. The digest is analyzed using the twice-running capability of an instrument that acquires a full-scan mass spectrum to determine the molecular weight of the peptide and a product ion spectrum to determine the amino acid sequence in a continuous scan. analyzed. With this aspect of the analysis,
A range of amounts over several orders of magnitude yields about 400 CAD spectrum ions. Not all CAD spectra are derived from peptides.

[0098] Data was analyzed by database search using the SEQUEST search algorithm. Peptides that did not match by this algorithm were manually interpreted and searched against the EST database using the SEQUEST algorithm.

(Results) The peptides shown in Table 3 were detected in band C48. None of these peptides matched any of the proteins or EST entries. This peptide appears to be completely new.

[0100]

[Table 3] ¹ I and L are indistinguishable by low energy CAD, but are deduced by database sequences. M (o) indicates oxidized M. C is C ^a
Unless stated as (acrylamide), it has been modified with carbamidomethyl. _ Indicates a single unknown residue. --- indicates an unknown number of unknown residues. The abbreviations are as follows: “X” = Ile or Leu; XQQT indicates the following amino acid sequence: Ile Gln Gln Thr (SEQ ID NO: 41) and Leu Gln Gln Thr (SEQ ID NO: 42); The following amino acid sequence is shown: Ile Asn Ser Gln Trp Val Val Pro Leu
Arg (SEQ ID NO: 1); and Leu Asn Ser Gln Trp Val Val Pro Leu
Arg (SEQ ID NO: 2); MVNXMXXXR shows the following amino acid sequence; Met Val Asn Ile Met Ile Ile Ile Arg (
SEQ ID NO: 3), Met Val Asn Leu Met Ile Ile Ile Arg (
SEQ ID NO: 4), Met Val Asn Ile Met Leu Ile Ile Arg (
SEQ ID NO: 5), Met Val Asn Ile Met Ile Leu Ile Arg (
SEQ ID NO: 6), Met Val Asn Ile Met Ile Ile Leu Arg (
SEQ ID NO: 7), Met Val Asn Ile Met Ile Leu Leu Arg (
SEQ ID NO: 8), Met Val Asn Ile Met Leu Leu Ile Arg (
SEQ ID NO: 9), Met Val Asn Ile Met Leu Ile Leu Arg (
SEQ ID NO: 10), Met Val Asn Leu Met Leu Ile Ile Arg (
SEQ ID NO: 11), Met Val Asn Leu Met Leu Ile Ile Arg (
SEQ ID NO: 12), Met Val Asn Leu Met Ile Leu Ile Arg (
SEQ ID NO: 13), Met Val Asn Leu Met Ile Ile Leu Arg (
SEQ ID NO: 14), Met Val Asn Leu Met Ile Leu Leu Arg (
SEQ ID NO: 15), Met Val Asn Leu Met Leu Leu Ile Arg (
SEQ ID NO: 16), Met Val Asn Leu Met Leu Ile Leu Arg (
SEQ ID NO: 17).

Example 4 Peptides from Band 2 were isolated as described in Examples 3 and 4. The peptides shown in Table 4 were detected in the band 2 digest. And this could not match any database sequence. The peptides were not very abundant, and the digest contained a number of more abundant peptides, all of which matched the keratin sequence.

[0102]

[Table 4] ¹ I and L are indistinguishable by low energy CAD, but are deduced by database sequences. Mo indicates oxidized M. C is modified with carbamidomethyl unless otherwise stated. _ Indicates a single unknown residue. --- indicates an unknown number of unknown residues. The molecular weight for the ² + 2 ion is the monoisotopic molecular weight, and for the> +3 ion is the average molecular weight. The abbreviations are as follows: "X
"= Ile or Leu; PXSS indicates the following amino acid sequence: Pro Ile Ser Ser (SEQ ID NO: 43), and Pro Leu Ser Ser (SEQ ID NO: 44); MXPVXE indicates the following amino acid sequence: Met Ile Pro Val Ile Glu (SEQ ID NO: 18) Met Leu Pro Val Ile Glu (SEQ ID NO: 19) Met Ile Pro Val Leu Glu (SEQ ID NO: 20), and Met Leu Pro Val Ile Glu (SEQ ID NO: 21); The sequence is shown: His Gly Gln Ser Ala Glu Tyr Glu Phe (
SEQ ID NO: 22).

FQQDGGAS shows the following amino acid sequence: Phe Gln Gln Asp Gly Gly Alya Ser (SEQ ID NO: 23).

Band 3. The peptides shown in Table 5 were detected in the band 3 digest. Analysis of this data was complicated because the digested protein was not very abundant. Peptides 4 and 6 were identified in the database sequence of the human B7 protein (
NCBI. nr. 01.04.99 registration number 1732415, calculated MW = 3
6.1 kDa, calculated pI 5.0). Peptide 3 was converted to bacillus I
Type of signal peptidase in the database sequence (NCBI.nr.
01.04.99 registration number 126186, calculated MW = 21.0 kDa, calculated pI 9.4). No matching database was found for any other peptides, and not all of these peptides could match the EST database. We also found that both peptides 8 and 9 in band 3 were in band 2
Note that it is the same as peptides 2 and 3 in.

[0105]

[Table 5] ¹ I and L are indistinguishable by low energy CAD, but are deduced by database sequences. Mo indicates oxidized M. C is modified with carbamidomethyl unless otherwise stated. _ Indicates a single unknown residue. --- indicates an unknown number of unknown residues. The molecular weight for ² +2 ions is the molecular weight of a single isotope, and the molecular weight for> +3 ions is the average molecular weight. The abbreviations are as follows: "X" = Ile or Leu; YIGEFDR indicates the following amino acid sequence; Tyr Ile Gly Glu Phe Asp Arg (SEQ ID NO: 45); SLQYLNLR indicates the following amino acid sequence; Ser Leu Gln Tyr Leu Asn Leu Arg (SEQ ID NO: 46); DNQIDTSLGFSR shows the following amino acid sequence; Asp Asn Gln Ile Asp Thr Ser Leu Gly
MXPVXE shows the following amino acid sequence: Met Ile Pro Val Ile Glu (SEQ ID NO: 18), Met Leu Pro Val Ile Glu (SEQ ID NO: 19), Met Ile Pro Val Luu Glu (SEQ ID NO: 47); SEQ ID NO: 20), and Met Leu Pro Val Ile Glu (SEQ ID NO: 21); HGQSAEYEF shows the following amino acid sequence; His Gly Gln Ser Ala Glu Tyr Glu Phe (
SEQ ID NO: 22).

[Sequence list]

[Brief description of the drawings]

1A-1D compare five sera from fertile males (FIG. 1A) and fertile females (FIG. 1B) with five sterile males (FIG. 1C) and sterile females (FIG. 1D). Western blot analysis by serial incubation of simple blots (dilution 1:
2000). Major autoantigens and isoantigens (arrows) are recognized that are uniquely recognized by infertile subjects.

FIGS. 2A-2B represent computer generated images showing major autoantigens (FIG. 2A) and isoantigens (FIG. 2B) recognized by infertile serum. Computer-scanned images of Western blots were compared between fertile and infertile groups using a common protein spot across the two images as an anchor. Protein spots recognized by the serum of fertile subjects (both male and female) were subtracted from images representing sterile male and female subjects, and sterile male (FIG. 2A) and sterile female (
Images representing all antigens uniquely recognized by FIG. 2B) were generated. It is observed that there are more autoantigens than isoantigens and different patterns of immunoreactivity exist between the sexes. Arrows indicate protein spots recognized by both male and female subjects.

FIG. 3 depicts a computer-generated composite image of a two-dimensional IEF / PAGE vectorized iodinated human sperm protein. The spermatozoa were radiolabeled and lysed and separated by two-dimensional electrophoresis by IEF / PAGE as described in Example 1. This composite image was generated after scanning an autoradiographic image of surface proteins immobilized on a nitrocellulose membrane and comparing the image to the location of the autoantigen and isoantigen (FIG. 1). Arrows indicate the location of autoantigens and isoantigens on the immunodominant surface.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 15/16 A61P 15/18 15/18 C07K 16/28 C07K 16/28 G01N 33/15 Z G01N 33 / 15 33/50 J 33/50 Z 33/53 D 33/53 33/543 501A 33/543 501 C07K 14/705 // C07K 14/705 A61K 37/02 (81) Designated countries EP (AT, BE, CH , CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN) , GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, Z, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Nerby-Hansen, Sohren United Kingdom NND7 1D E London, Grants Close 70 (72) Inventor Flickinger, Charles Jay. United States Virginia 22903, Charlottesville, Meadowbrook Road 2009 (72) Inventor Shetty, Jagaspalla United States Virginia 22903, Charlottesville, Payton Court 287 F-term (reference) 2G045 AA27 AA40 CA25 CA26 DA36 FB03 FB07 4C084 AA02 BA17 CA36 MA66 NA14 ZA81 4C085 AA03 AA14 BB11 CC13 DD32 DD62 EE01 EE06 FF24 GG02 4H045 AA11 AA30 BA14 BA15 CA40 DA75 DA86 EA20 EA54 FA74

Claims (14)

[Claims] 1. An isolated or recombinant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 22, and SEQ ID NO: 23. 2. The method according to claim 1, wherein the amino acid sequence is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 18.
The polypeptide of claim 1, wherein the polypeptide is selected from the group consisting of: SEQ ID NO: 22 and SEQ ID NO: 23. 3. A method of diagnosing immunological infertility comprising the steps of: obtaining a serum sample from an individual; contacting the sample with a composition containing a sperm surface antigen; Screening for antigen / antibody reaction. 4. The method of claim 3, wherein screening for an antigen / antibody reaction comprises using a labeled secondary antibody. 5. The sperm surface antigen is bound to a solid substrate.
The method described in. 6. The sperm surface antigen is a protein recognized by serum isolated from an infertile patient, wherein the protein is not recognized by serum from a fertile patient. Item 6. The method according to Item 5. 7. The sperm surface antigen is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 22, and 23.
The method of claim 5. 8. A pharmaceutical composition comprising a peptide and a pharmaceutically acceptable carrier, wherein the peptide comprises SEQ ID NO: 1 to SEQ ID NO: 22, and SEQ ID NO: 23.
A pharmaceutical composition comprising an amino acid sequence selected from the group consisting of: 9. The pharmaceutical composition according to claim 8, further comprising an adjuvant. 10. A mammal, comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 22, and SEQ ID NO: 23. A method for modulating fertility in a mammalian species, comprising administering a composition that comprises: 11. The method of claim 10, wherein said composition further comprises an adjuvant. 12. The composition of claim 10, wherein the composition is administered by intravenous injection.
The method described in. 13. An antibody that immunospecifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 22, and SEQ ID NO: 23. 14. A contraceptive composition comprising the antibody according to claim 13 and a pharmaceutical carrier.